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Combination simvastatin and metformin induces G1-phase cell cycle arrest and Ripk1- and Ripk3-dependent necrosis in C4-2B osseous metastatic castration-resistant prostate cancer cells.

Babcook MA, Sramkoski RM, Fujioka H, Daneshgari F, Almasan A, Shukla S, Nanavaty RR, Gupta S - Cell Death Dis (2014)

Bottom Line: SIM+MET treatment led to enhanced autophagic flux in C4-2B cells by 72-96 h, ascertained by increased LC3B-II (further enhanced with lysosomal inhibitor chloroquine) and reduced Sequestosome-1 protein expression, significantly increased percentage of acidic vesicular organelle-positive cells, and increased autophagic structure accumulation assessed by transmission electron microscopy.Instead, SIM+MET treatment led to Ripk1- and Ripk3-dependent necrosis by 48-96 h, determined by propidium iodide-Annexin V flow cytometry, increase in Ripk1 and Ripk3 protein expression, necrosome formation, HMGB-1 extracellular release, and necrotic induction and viability rescue with necrostatin-1 and Ripk3-targeting siRNA.The necrosis-inducing capacity of SIM+MET may make these drugs a highly-effective treatment for apoptosis- and chemotherapy-resistant metastatic CRPC cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Nutrition, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA [2] Department of Urology, Case Western Reserve University School of Medicine & The Urology Institute, University Hospitals Case Medical Center, Cleveland, OH 44106, USA.

ABSTRACT
Castration-resistant prostate cancer (CRPC) cells acquire resistance to chemotherapy and apoptosis, in part, due to enhanced aerobic glycolysis and biomass production, known as the Warburg effect. We previously demonstrated that combination simvastatin (SIM) and metformin (MET) ameliorates critical Warburg effect-related metabolic aberrations of C4-2B cells, synergistically and significantly decreases CRPC cell viability and metastatic properties, with minimal effect on normal prostate epithelial cells, and inhibits primary prostate tumor growth, metastasis, and biochemical failure in an orthotopic model of metastatic CRPC, more effectively than docetaxel chemotherapy. Several modes of cell death activated by individual treatment of SIM or MET have been reported; however, the cell death process induced by combination SIM and MET treatment in metastatic CRPC cells remains unknown. This must be determined prior to advancing combination SIM and MET to clinical trial for metastatic CRPC. Treatment of C4-2B cells with combination 4 μM SIM and 2 mM MET (SIM+MET) led to significant G1-phase cell cycle arrest and decrease in the percentage of DNA-replicating cells in the S-phase by 24 h; arrest was sustained throughout the 96-h treatment. SIM+MET treatment led to enhanced autophagic flux in C4-2B cells by 72-96 h, ascertained by increased LC3B-II (further enhanced with lysosomal inhibitor chloroquine) and reduced Sequestosome-1 protein expression, significantly increased percentage of acidic vesicular organelle-positive cells, and increased autophagic structure accumulation assessed by transmission electron microscopy. Chloroquine, however, could not rescue CRPC cell viability, eliminating autophagic cell death; rather, autophagy was upregulated by C4-2B cells in attempt to withstand chemotherapy. Instead, SIM+MET treatment led to Ripk1- and Ripk3-dependent necrosis by 48-96 h, determined by propidium iodide-Annexin V flow cytometry, increase in Ripk1 and Ripk3 protein expression, necrosome formation, HMGB-1 extracellular release, and necrotic induction and viability rescue with necrostatin-1 and Ripk3-targeting siRNA. The necrosis-inducing capacity of SIM+MET may make these drugs a highly-effective treatment for apoptosis- and chemotherapy-resistant metastatic CRPC cells.

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Combination simvastatin and metformin treatment does not induce apoptosis in C4-2B metastatic CRPC cells. (a) Western blot analysis of total and cleaved caspase-3 and cleaved PARP protein expression from total cell lysates of C4-2B3 and C4-2B4 cells following treatment with 4 μM simvastatin (SIM) and/or 2 mM metformin (MET) for 24−72 h. Total cell lysates of C4-2B3 and C4-2B4 cells treated with 2 μM (S)-(+)-camptothecin (CAMP) for 48 h used as apoptosis positive control. GAPDH used as loading control. (b) C4-2B3 and C4-2B4 cells treated with 4 μM SIM and/or 2 mM MET for 48−96 h followed by staining with FITC-conjugated Annexin V (AV) and propidium iodide (PI) and analyzed by flow cytometry. Percentage of PI(−)AV(−), PI(−)AV(+), PI(+)AV(−), and PI(+)AV(+) cells demonstrated in bar graphs of mean±S.D. from triplicate samples. Representative density plots of controls and samples shown in Supplementary Figure S1. (c) Percentage cell viability (mean±S.D.) by methylene blue assay of C4-2B3 and C4-2B4 cells following treatment with combination 4 μM SIM and 2 mM MET±10 μM z-VAD-fmk pan-caspase inhibitor for 24−96 h, n=3 per treatment group. NS denotes not significant difference in cell viability between the treatment groups of SIM+MET in presence or absence of z-VAD-fmk as determined by the two-tailed Student's t-test
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fig3: Combination simvastatin and metformin treatment does not induce apoptosis in C4-2B metastatic CRPC cells. (a) Western blot analysis of total and cleaved caspase-3 and cleaved PARP protein expression from total cell lysates of C4-2B3 and C4-2B4 cells following treatment with 4 μM simvastatin (SIM) and/or 2 mM metformin (MET) for 24−72 h. Total cell lysates of C4-2B3 and C4-2B4 cells treated with 2 μM (S)-(+)-camptothecin (CAMP) for 48 h used as apoptosis positive control. GAPDH used as loading control. (b) C4-2B3 and C4-2B4 cells treated with 4 μM SIM and/or 2 mM MET for 48−96 h followed by staining with FITC-conjugated Annexin V (AV) and propidium iodide (PI) and analyzed by flow cytometry. Percentage of PI(−)AV(−), PI(−)AV(+), PI(+)AV(−), and PI(+)AV(+) cells demonstrated in bar graphs of mean±S.D. from triplicate samples. Representative density plots of controls and samples shown in Supplementary Figure S1. (c) Percentage cell viability (mean±S.D.) by methylene blue assay of C4-2B3 and C4-2B4 cells following treatment with combination 4 μM SIM and 2 mM MET±10 μM z-VAD-fmk pan-caspase inhibitor for 24−96 h, n=3 per treatment group. NS denotes not significant difference in cell viability between the treatment groups of SIM+MET in presence or absence of z-VAD-fmk as determined by the two-tailed Student's t-test

Mentions: Apoptosis may be caused by toxins or nutrient deprivation and is characterized by cellular blebbing, cytoplasmic and nuclear shrinkage, chromatin condensation, and DNA fragmentation.22, 23 The spike (5.0–7.3%) of sub-G1 cells noted at 48-h SIM+MET treatment (Figure 2), indicative of DNA fragmentation, suggests some C4-2B cells may be undergoing apoptosis at this time point. Therefore, we evaluated the potential of SIM+MET to induce cell death by apoptosis using methods to capture potential activation of mitochondrial (intrinsic) and/or death receptor signaling (extrinsic) apoptotic pathways. Caspase-3 and downstream poly-(ADP-ribose) polymerase (PARP) are cleaved and activated by both pathways of apoptosis.24 In distinct contrast to apoptosis-inducing positive control (S)-(+)-camptothecin treatment, neither caspase-3 nor PARP cleavage was noted following SIM+MET treatment for 24–72 h (Figure 3a).


Combination simvastatin and metformin induces G1-phase cell cycle arrest and Ripk1- and Ripk3-dependent necrosis in C4-2B osseous metastatic castration-resistant prostate cancer cells.

Babcook MA, Sramkoski RM, Fujioka H, Daneshgari F, Almasan A, Shukla S, Nanavaty RR, Gupta S - Cell Death Dis (2014)

Combination simvastatin and metformin treatment does not induce apoptosis in C4-2B metastatic CRPC cells. (a) Western blot analysis of total and cleaved caspase-3 and cleaved PARP protein expression from total cell lysates of C4-2B3 and C4-2B4 cells following treatment with 4 μM simvastatin (SIM) and/or 2 mM metformin (MET) for 24−72 h. Total cell lysates of C4-2B3 and C4-2B4 cells treated with 2 μM (S)-(+)-camptothecin (CAMP) for 48 h used as apoptosis positive control. GAPDH used as loading control. (b) C4-2B3 and C4-2B4 cells treated with 4 μM SIM and/or 2 mM MET for 48−96 h followed by staining with FITC-conjugated Annexin V (AV) and propidium iodide (PI) and analyzed by flow cytometry. Percentage of PI(−)AV(−), PI(−)AV(+), PI(+)AV(−), and PI(+)AV(+) cells demonstrated in bar graphs of mean±S.D. from triplicate samples. Representative density plots of controls and samples shown in Supplementary Figure S1. (c) Percentage cell viability (mean±S.D.) by methylene blue assay of C4-2B3 and C4-2B4 cells following treatment with combination 4 μM SIM and 2 mM MET±10 μM z-VAD-fmk pan-caspase inhibitor for 24−96 h, n=3 per treatment group. NS denotes not significant difference in cell viability between the treatment groups of SIM+MET in presence or absence of z-VAD-fmk as determined by the two-tailed Student's t-test
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Combination simvastatin and metformin treatment does not induce apoptosis in C4-2B metastatic CRPC cells. (a) Western blot analysis of total and cleaved caspase-3 and cleaved PARP protein expression from total cell lysates of C4-2B3 and C4-2B4 cells following treatment with 4 μM simvastatin (SIM) and/or 2 mM metformin (MET) for 24−72 h. Total cell lysates of C4-2B3 and C4-2B4 cells treated with 2 μM (S)-(+)-camptothecin (CAMP) for 48 h used as apoptosis positive control. GAPDH used as loading control. (b) C4-2B3 and C4-2B4 cells treated with 4 μM SIM and/or 2 mM MET for 48−96 h followed by staining with FITC-conjugated Annexin V (AV) and propidium iodide (PI) and analyzed by flow cytometry. Percentage of PI(−)AV(−), PI(−)AV(+), PI(+)AV(−), and PI(+)AV(+) cells demonstrated in bar graphs of mean±S.D. from triplicate samples. Representative density plots of controls and samples shown in Supplementary Figure S1. (c) Percentage cell viability (mean±S.D.) by methylene blue assay of C4-2B3 and C4-2B4 cells following treatment with combination 4 μM SIM and 2 mM MET±10 μM z-VAD-fmk pan-caspase inhibitor for 24−96 h, n=3 per treatment group. NS denotes not significant difference in cell viability between the treatment groups of SIM+MET in presence or absence of z-VAD-fmk as determined by the two-tailed Student's t-test
Mentions: Apoptosis may be caused by toxins or nutrient deprivation and is characterized by cellular blebbing, cytoplasmic and nuclear shrinkage, chromatin condensation, and DNA fragmentation.22, 23 The spike (5.0–7.3%) of sub-G1 cells noted at 48-h SIM+MET treatment (Figure 2), indicative of DNA fragmentation, suggests some C4-2B cells may be undergoing apoptosis at this time point. Therefore, we evaluated the potential of SIM+MET to induce cell death by apoptosis using methods to capture potential activation of mitochondrial (intrinsic) and/or death receptor signaling (extrinsic) apoptotic pathways. Caspase-3 and downstream poly-(ADP-ribose) polymerase (PARP) are cleaved and activated by both pathways of apoptosis.24 In distinct contrast to apoptosis-inducing positive control (S)-(+)-camptothecin treatment, neither caspase-3 nor PARP cleavage was noted following SIM+MET treatment for 24–72 h (Figure 3a).

Bottom Line: SIM+MET treatment led to enhanced autophagic flux in C4-2B cells by 72-96 h, ascertained by increased LC3B-II (further enhanced with lysosomal inhibitor chloroquine) and reduced Sequestosome-1 protein expression, significantly increased percentage of acidic vesicular organelle-positive cells, and increased autophagic structure accumulation assessed by transmission electron microscopy.Instead, SIM+MET treatment led to Ripk1- and Ripk3-dependent necrosis by 48-96 h, determined by propidium iodide-Annexin V flow cytometry, increase in Ripk1 and Ripk3 protein expression, necrosome formation, HMGB-1 extracellular release, and necrotic induction and viability rescue with necrostatin-1 and Ripk3-targeting siRNA.The necrosis-inducing capacity of SIM+MET may make these drugs a highly-effective treatment for apoptosis- and chemotherapy-resistant metastatic CRPC cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Nutrition, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA [2] Department of Urology, Case Western Reserve University School of Medicine & The Urology Institute, University Hospitals Case Medical Center, Cleveland, OH 44106, USA.

ABSTRACT
Castration-resistant prostate cancer (CRPC) cells acquire resistance to chemotherapy and apoptosis, in part, due to enhanced aerobic glycolysis and biomass production, known as the Warburg effect. We previously demonstrated that combination simvastatin (SIM) and metformin (MET) ameliorates critical Warburg effect-related metabolic aberrations of C4-2B cells, synergistically and significantly decreases CRPC cell viability and metastatic properties, with minimal effect on normal prostate epithelial cells, and inhibits primary prostate tumor growth, metastasis, and biochemical failure in an orthotopic model of metastatic CRPC, more effectively than docetaxel chemotherapy. Several modes of cell death activated by individual treatment of SIM or MET have been reported; however, the cell death process induced by combination SIM and MET treatment in metastatic CRPC cells remains unknown. This must be determined prior to advancing combination SIM and MET to clinical trial for metastatic CRPC. Treatment of C4-2B cells with combination 4 μM SIM and 2 mM MET (SIM+MET) led to significant G1-phase cell cycle arrest and decrease in the percentage of DNA-replicating cells in the S-phase by 24 h; arrest was sustained throughout the 96-h treatment. SIM+MET treatment led to enhanced autophagic flux in C4-2B cells by 72-96 h, ascertained by increased LC3B-II (further enhanced with lysosomal inhibitor chloroquine) and reduced Sequestosome-1 protein expression, significantly increased percentage of acidic vesicular organelle-positive cells, and increased autophagic structure accumulation assessed by transmission electron microscopy. Chloroquine, however, could not rescue CRPC cell viability, eliminating autophagic cell death; rather, autophagy was upregulated by C4-2B cells in attempt to withstand chemotherapy. Instead, SIM+MET treatment led to Ripk1- and Ripk3-dependent necrosis by 48-96 h, determined by propidium iodide-Annexin V flow cytometry, increase in Ripk1 and Ripk3 protein expression, necrosome formation, HMGB-1 extracellular release, and necrotic induction and viability rescue with necrostatin-1 and Ripk3-targeting siRNA. The necrosis-inducing capacity of SIM+MET may make these drugs a highly-effective treatment for apoptosis- and chemotherapy-resistant metastatic CRPC cells.

Show MeSH
Related in: MedlinePlus